The Immunodeficiency, Centromeric instability, and Facial anomalies (ICF) syndrome is a rare autosomal recessive disorder, with the vast majority of cases carrying mutations in either the DNA methyltransferase gene DNMT3B (ICF1) or the zinc finger protein gene ZBTB24 (ICF2). A hallmark of ICF syndrome is loss of DNA methylation in specific genomic regions, which is believed to be the primary defect underlying other phenotypic abnormalities, including antibody deficiency (hypogammaglobulinemia), facial dysmorphism, and mental retardation. Patients with ICF syndrome usually die of recurrent infections in early childhood. Although ICF syndrome was first reported nearly four decades ago, little progress has been made in understanding the pathogenesis of the disease, largely because of the lack of appropriate in vitro and in vivo models. Modeling ICF syndrome using Dnmt3b mutant mice has been a challenge, because complete inactivation of Dnmt3b leads to embryonic lethality and mice carrying ICF-like point mutations fail to recapitulate antibody deficiency. Preliminary data from the applicant's laboratory revealed that Zbtb24 depletion in mouse embryonic stem cells (ESCs) results in substantial Dnmt3b downregulation and DNA methylation alterations characteristic of ICF syndrome and that conditional Zbtb24 deletion in the hematopoietic lineage leads to severe hypogammaglobulinemia in mice, apparently due to defects in plasma cell differentiation or survival. The objective of this application is to determine the role of Zbtb24 in the regulation of DNA methylation and antibody production. The central hypothesis is that Zbtb24, via regulating Dnmt3b expression, controls DNA methylation, gene expression, and chromatin structure in lymphocyte populations that are important for antibody production. The applicant proposes to use Zbtb24-deficient ESCs to elucidate the molecular mechanism by which Zbtb24 regulates Dnmt3b expression and determine the impacts of Zbtb24 deficiency on DNA methylation, gene expression, and chromatin structure (aim 1). The applicant also proposes to use the Zbtb24-mutant ICF mouse model to determine the cellular defects involved in antibody deficiency and elucidate the links between aberrant DNA methylation and the defects in gene expression, chromatin structure and molecular signaling that contribute to antibody deficiency (aim 2). The project is significant, because results from the proposed work will not only fundamentally advance mechanistic understanding of ICF syndrome, but could also shed light on the etiology and defects underlying other antibody deficiency diseases, including common variable immunodeficiency (CVID). These results will also have general implications in understanding the role of epigenetic mechanisms in immunity and immunological disorders. The project has potential translational impact as well, because results from the proposed work could lead to identification of novel therapeutic strategies for ICF syndrome and other immunodeficiency diseases, and the innovative ICF mouse model could be valuable for preclinical testing of therapeutics in the future.